Modeling the Origins of Lymphoid Blast Crisis Via Expression of BCR/ABL in Hematopoietic Cells Mutated at the INK4a/ARF Locus.

Abstract

Homozygous deletion of exon 2 at the INK4a/ARF locus is found in approximately 50% of BCR/ABL-mediated lymphoid blast crisis specimens, indicating that dysfunction of the locus is a contributing factor in lymphoid leukemogenesis. The INK4a/ARF locus encodes tumor suppressors, p16INK4a and p14 (human)/p19 (mouse) ARF, which are involved in regulating the Rb-mediated G1 cell cycle check point and MDM2-mediated p53 degradation respectively. Here we report a novel murine acute lymphoblastic leukemia (ALL) model which creates a disease similar to human B-ALL. In this system, we used a retrovirus encoding BCR/ABL-p210 and green fluorescent protein (GFP) to transduce INK4a/ARF null hematopoietic cells (exon 2 deleted), followed by transplantation into wild-type C57Bl/6 recipient mice. As early as 10–12 days post-transplant, transduced GFP+ cells were detected in the peripheral circulation. By 14–16 days post-transplant, recipient mice had highly elevated white blood cell counts and signs of advanced disease (hunched posture, roughened coat, anorexia and inactivity). Flow cytometric studies indicated that peripheral GFP+ cells were almost entirely B lymphoid (B220 and CD19+), and cytological analysis showed a blast-like morphology. In addition, the disease was readily transferred to secondary and tertiary recipients by bone marrow transplantation. These findings indicated rapid and reproducible development of B-ALL in recipient animals. To characterize potential leukemic progenitor cells, in vitro colony assays were performed using marrow, spleen and peripheral blood cells. These experiments showed that almost all in vitro colony-forming ability resided in marrow and represented approximately 2% of the overall leukemic population. Further, the phenotype of colony-forming leukemic cells correlated with expression of VLA5 and VCAM-1. In order to explore potential target cells responsible for the disease phenotype, different subpopulations of donor INK4a/ARF null hematopoietic cells were sorted, transduced with the BCR/ABL-p210 retroviral vector, and transplanted into wild type recipients. These experiments demonstrated that B-ALL could arise from BCR/ABL expression in INK4a/ARF null hematopoietic stem cells (HSC) and Pro-B cells, but not from common myeloid progenitors (CMP) or IgM+B cells. Interestingly, while transplantation of infected Pro-B cells generated only B-ALL, the HSC-transduced recipient mice showed mixed lineage engraftment with contribution to both myeloid and lymphoid tissues. In these animals lymphoid GFP+ cells showed the same immature B-ALL phenotype as observed previously, while myeloid GFP+ cells showed heterogeneous myeloid subsets, consistent with a CML-like myeloproliferative disease. Over time, the B-ALL population in these mice progressed rapidly and the animals succumbed to ALL. Subsequent transplantation analysis of marrow from mixed lineage animals demonstrated that both the B-ALL and myeloid phenotypes could be transferred to secondary recipients. These findings indicate that leukemic stem cells (LSC) are detectable in this model of lymphoid blast crisis, but that the clinical presentation is highly dependent upon the developmental stage at which mutations occur. Given the typical myeloid phenotype observed upon expression of the BCR/ABL retroviral vector in wild type HSC, our findings indicate that INK4a/ARF function critically influences developmental fate choices in the progression of BCR/ABL-mediated leukemia from chronic to blast crisis phase.